Expression of priming-associated cellular markers on neutrophils during an exacerbation of COPD.

Chronic inflammation of the airways is a hallmark of chronic obstructive pulmonary disease (COPD). We investigated the kinetics of priming of inflammatory cells in peripheral blood during exacerbations of COPD and during the resolution phase. Modulation of the leukocyte compartment as a consequence of systemic activation by cytokines/chemokines was determined by measuring the expression of priming-associated epitopes by novel antibodies designated A17 and A27. Furthermore, H2O2 was determined in breath condensate as a read out for local inflammation. Leukocytes were obtained from COPD patients (GOLD II-IV) during and after an exacerbation of their disease. During an exacerbation the expression of priming epitopes on leukocytes was increased. This priming phenotype disappeared upon treatment with intravenous corticosteroids. Similarly, H2O2 levels in breath condensate were also increased during an exacerbation and decreased upon treatment. We conclude that the activation status of neutrophils in the systemic compartment can be used as a read-out for systemic innate immune signals involved in the pathogenesis of COPD. The correlation between H2O2 in exhaled air with A27 priming on neutrophils showed that local inflammation has systemic effects on cells of the innate immune system.

Differential expression of genes involved in skin homing, proliferation, and apoptosis in CD4+ T cells of patients with atopic dermatitis.

CD4+ T cells play a critical role in allergic diseases, both in the affected tissue as well as systemically. Our objective was to investigate the in vivo activation state of peripheral blood CD4+ T cells of atopic dermatitis (AD) patients by analyzing gene expression profiles of unstimulated CD4+ T cells. mRNA samples from blood CD4+ T cells, isolated from five AD patients and seven healthy controls (HC), were analyzed using oligonucleotide arrays. Differentially regulated genes were validated by quantitative PCR (Q-PCR) in a larger group of patients with AD, in a group of patients with allergic asthma (AA), and HC subjects. In addition, "typical" T helper type 1 (Th1)- and Th2-related genes were analyzed by Q-PCR. Microarray analysis revealed differential expression of 52 genes in AD patients. Q-PCR confirmed several differentially regulated genes in AD, including CCR10, CRTH2, C-JUN, and NR4A2. Two groups of genes with highly correlating gene expression levels involved in tissue homing and proliferation or apoptosis, respectively, were identified. No marked differences were found in gene expression levels of typical Th1 or Th2 genes in AD or in AA patients. This study demonstrates that peripheral blood, unstimulated CD4+ T cells in AD patients show differentially expressed genes involved in tissue homing, proliferation, and apoptosis. No marked expression differences of "typical" atopy genes were found.

Geranylgeranylated proteins are involved in the regulation of myeloma cell growth.

PURPOSE: Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. This study examined the role of farnesylated and geranylgeranylated proteins in the regulation of myeloma cell proliferation. EXPERIMENTAL DESIGN: Antiproliferative and apoptotic effects of various modulators of farnesylated and geranylgeranylated proteins were investigated in myeloma cells. RESULTS: Depletion of geranylgeranylpyrophosphate inhibited myeloma cell proliferation through accumulation of cells in G(1) phase of the cell cycle and loss of cells in S phase. In contrast, depletion of farnesylpyrophosphate had no or only minor effects. Furthermore, inhibition of geranylgeranyl transferase I activity was more effective in reducing myeloma cell growth when compared with inhibition of farnesyl transferase activity. This indicates that protein geranylgeranylation is important for myeloma cell proliferation and cell cycle progression through G(1). Geranylgeranylated target proteins involved in the control of proliferation include GTPases, such as Rac-1, Cdc42, and RhoA. Inhibition of Rho, Rac, and Cdc42 GTPases by toxin B reduced proliferation, without affecting cell viability, whereas specific inhibition of Rho GTPases by C3 exoenzyme was without effect. This suggests a role for Rac and/or Cdc42 GTPases in myeloma cell growth. Rac-1 activity was found in all myeloma cell lines and was suppressed by the depletion of intracellular pools of geranylgeranylpyrophosphate, whereas interleukin-6 rapidly induced Rac-1 activation. Furthermore, dominant-negative Tat-Rac-1 reduced myeloma cell proliferation, whereas constitutively active Tat-Rac-1 enhanced proliferation. CONCLUSION: These results indicate that protein geranylgeranylation is essential for myeloma cell proliferation and suggest that Rac-1 is a regulator of myeloma cell growth.

Decreased neutrophil skin infiltration after UVB exposure in patients with polymorphous light eruption.

UV radiation, in particular UVB, suppresses the skin immune response. In patients with polymorphous light eruption (PLE) the skin immune response seems activated after UV exposure. Typical PLE skin lesions can occur as early as several hours after UV exposure. In healthy volunteers, neutrophils infiltrate the skin shortly after UV exposure. The kinetics and mechanisms of neutrophil infiltration in the skin of PLE patients after UVB exposure was studied. Skin biopsies at 0, 3, 6, and 18 h were taken from five PLE patients and six healthy controls after irradiation with three minimal erythema dose UVB. Furthermore, neutrophils were isolated from blood of five PLE patients and six healthy controls to test their chemotactic activity. Immunohistochemical analysis showed a significant decreased neutrophil infiltration in PLE skin after UVB irradiation compared with healthy controls (p<0.05). In both healthy controls and PLE patients, after UVB irradiation, ICAM-1 and E-selectin expression on endothelial cells increased at 6 h after irradiation. Blood neutrophil chemotactic response towards IL-8 and C5a, as well as the expression of cell surface markers involved in adhesion and chemotaxis, was not different between PLE patients and healthy controls. In conclusion, PLE is marked by a decreased skin infiltration of neutrophils after UVB irradiation, possibly leading to a diminished neutrophil-induced suppression.

Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning.

Tyrosine phosphorylation is thought to be critical in the regulation of neutrophil functioning, and members of the Src family of tyrosine kinases have recently been shown to be regulated in activated granulocytes. We have used a specific pharmacological inhibitor of Src kinases, pyrazolpyrimidine 1 (PP1), to evaluate the role of Src kinases in cytokine/chemoattractant-induced regulation of neutrophil function. PP1 inhibits PKB phosphorylation but not STAT5 phosphorylation or the activation of MAP kinases by fMLP or GM-CSF. Pretreatment of neutrophils with PP1 and with the PI3K inhibitor LY294002 resulted in a strong inhibition of fMLP-induced superoxide production and cytokine-mediated survival but not fMLP-induced migration. It is interesting that the kinetics of inhibition of actin polymerization and the respiratory burst are very similar. Although initiation of both processes was not affected, sustained activation was inhibited by PP1. Taken together, our results demonstrate a critical role for Src kinases in regulating neutrophil cytotoxic-effector functioning through PI3K-PKB.